Sequence of rat alpha- and gamma-casein mRNAs: evolutionary comparison of the calcium-dependent rat casein multigene family.

The complete sequences of rat alpha- and gamma-casein mRNAs have been determined. The 1402-nucleotide alpha- and 864-nucleotide gamma-casein mRNAs both encode 15 amino acid signal peptides and mature proteins of 269 and 164 residues, respectively. Considerable homology between the 5' non-coding regions, and the regions encoding the signal peptides and the phosphorylation sites, in these mRNAs as compared to several other rodent casein mRNAs, was observed. Significant homology was also detected between rat alpha- and bovine alpha s1-casein. Comparison of the rodent and bovine sequences suggests that the caseins evolved at about the time of the appearance of the primitive mammals. This may have occurred by intragenic duplication of a nucleotide sequence encoding a primitive phosphorylation site, -(Ser)n-Glu-Glu-, and intergenic duplication resulting in the small casein multigene family. A unique feature of the rat alpha-casein sequence is an insertion in the coding region containing 10 repeated elements of 18 nucleotides each. This insertion appears to have occurred 7-12 million years ago, just prior to the divergence of rat and mouse.     

Genomic structure and evolution of the human pepsinogen A multigene family

Summary A human cosmid library was screened with a pepsinogen A (PGA) cDNA probe, yielding 18 clones with (parts of) one, two or three PGA genes. By aligning these cosmids a restriction map of a PGA gene quadruplet was obtained in which the four genes are arranged in a highly ordered fashion in a head-to-tail orientation. Using the length in kilobases of the large polymorphic EcoRI fragment of the PGA genes, this quadruplet can be described as 15.0-12.0-12.0-16.6. An AvaII polymorphism allowed us to identify the two PGA haplotypes of the individual whose DNA had been cloned in the cosmid library to be a gene triplet and a gene quadruplet. By comparing the restriction maps of the central 12.0 genes in these multiplets to those of the flanking 15.0 and 16.6 genes, we postulate that these central genes arose from unequal but homologous crossing over between two 15.0–16.6 gene pairs. This hypothesis provides for the creation of a variety of haplotypes by additional cross overs and mutations. Southern blots of family and population material supports the existance of at least five common PGA haplotypes, including a single-gene haplotype, giving rise to a large number of different EcoRI patterns. The single PGA gene is probably the reciprocal crossing over product. Comparison between the DNA and protein polymorphisms suggests further micro-heterogeneity in the different PGA haplotypes.     

The casein genes of Bos taurus. II. Isolation and characteristics of the beta-casein gene

The expression of the casein genes in mammary gland cells is regulated by peptide and steroid hormones. To study underlying regulatory mechanisms, the bovine beta-casein gene was isolated and characterized from lambda bacteriophage bovine DNA library. The beta-casein gene is 8.6 kb long and is 7.8 times longer than the mature casein mRNA coded for by 9 exons. The genomic clones incorporate additional 8.5 and 4.5 kb of the 5'- and 3'-flanking regions. The nucleotide sequences of 5' and 3' ends of the beta-casein gene are determined. Conserved sequences identical or homologous to potential sites of binding with the nuclear factor CTF/NF-1, glucocorticoid and progesterone receptors were identified. The regulatory region of the casein gene contains two different TATA signals flanking the duplication site in the promoter region.     

Gene expression of rat glutathione S-transferases. Evidence for gene conversion in the evolution of the Yb multigene family

We have characterized a cDNA with complete coding sequence for the rat liver glutathione S-transferase subunit 4 (Yb2) isolated from a constructed lambda gt10 cDNA library. Functional expression of the cDNA sequence has resulted in the purification to homogeneity of an enzymatically active anionic glutathione S-transferase. In addition to three previously described Yb-type subunits (Yb1, Yb2, Yb3), we now report characterization of a fourth Yb subunit sequence in the form of a genomic DNA clone lambda GTR15-2. The Yb4 gene has no apparent defect, and the deduced Yb4 polypeptide sequence differs from the other three Ybs by 40 to 53 amino acids. The Yb4 gene organization is similar to that of the Yb2 gene in having a minimum of eight exons. Three out of the seven introns between the two genes are conserved to the extent of more than 88% nucleotide identity. We propose that gene conversion may have played a role in the evolution of these Yb genes.     

Structure and evolution of the apolipoprotein multigene family

We present the complementary DNA and deduced amino acid sequence of rat apolipoprotein A-II (apoA-II), and the results of a detailed statistical analysis of the nucleotide and amino acid sequences of all the apolipoprotein gene sequences published to date: namely, those of human and rat apoA-I, apoA-II and apoE, rat apoA-IV, and human apoC-I, C-II and C-III. Our results indicate that the apolipoprotein genes have very similar genomic structures, each having a total of three introns at the same locations. Using the exon/intron junctions as reference points, we have obtained an alignment of the coding regions of all the genes studied. It appears that the mature peptide regions of these genes are almost completely made up of tandem repeats of 11 codons. The part of mature peptide region encoded by exon 3 contains a common block of 33 codons, whereas the part encoded by exon 4 contains a much more variable number of internal repeats of 11 codons. These genes have apparently evolved from a primordial gene through multiple partial (internal) and complete gene duplications. On the basis of the degree of homology of the various sequences, and the pattern of the internal repeats in these genes, we propose an evolutionary tree for the apolipoprotein genes and give rough estimates of the divergence times between these genes. Our results show that apoA-II has evolved extremely rapidly and that apoA-I and apoE also have evolved at high rates but some regions are better conserved than the others. The rate of evolution of individual regions seems to be related to the stringency of their functional requirements.     

The olfactory multigene family.

A novel multigene family has been identified that is likely to encode odorant receptors on olfactory sensory neurons. Further studies on this gene family are likely to shed light on the molecular mechanisms underlying information coding in the mammalian olfactory system. This review is also published in Current Opinion in Genetics and Development 1992, 2:467-473.     

The olfactory multigene family.

A novel multigene family has been identified that is likely to encode odorant receptors on olfactory sensory neurons. Further studies on this gene family are likely to shed light on the molecular mechanisms underlying information coding in the mammalian olfactory system. This review is also published in Current Opinion in Neurobiology 1992, 2:282-288.     

Molecular evolution of the HSP70 multigene family

Eukaryotic genomes encode multiple 70-kDa heat-shock proteins (HSP70s). The Saccharomyces cerevisiae HSP70 family is comprised of eight members. Here we present the nucleotide sequence of the SSA3 and SSB2 genes, completing the nucleotide sequence data for the yeast HSP70 family. We have analyzed these yeast sequences as well as 29 HSP70s from 24 additional eukaryotic and prokaryotic species. Comparison of the sequences demonstrates the extreme conservation of HSP70s; proteins from the most distantly related species share at least 45% identity and more than one-sixth of the amino acids are identical in the aligned region (567 amino acids) among all proteins analyzed. Phylogenetic trees constructed by two independent methods indicate that ancient molecular and cellular events have given rise to at least four monophyletic groups of eukaryotic HSP70 proteins. Each group of evolutionarily similar HSP70s shares a common intracellular localization and is presumed to be comprised of functional homologues; these include heat-shock proteins of the cytoplasm, endoplasmic reticulum, mitochondria, and chloroplasts. HSP70s localized in mitochondria and plastids are most similar to the DnaK HSP70 homologues in purple bacteria and cyanobacteria, respectively, which is consistent with the proposed prokaryotic origin of these organelles. The analyses indicate that the major eukaryotic HSP70 groups arose prior to the divergence of the earliest eukaryotes, roughly 2 billion years ago. In some cases, as exemplified by the SSA genes encoding the cytoplasmic HSP70s of S. cerevisiae, more recent duplication events have given rise to subfamilies within the major groups. The S. cerevisiae SSB proteins comprise a unique subfamily not identified in other species to date. This subfamily appears to have resulted from an ancient gene duplication that occurred at approximately the same time as the origin of the major eukaryotic HSP70 groups. Correspondence to: E.A. Craig     

Exon-intron structure and evolution of the Lipocalin gene family

The Lipocalins are an ancient protein family whose expression is currently confirmed in bacteria, protoctists, plants, arthropods, and chordates. The evolution of this protein family has been assessed previously using amino acid sequence phylogenies. In this report we use an independent set of characters derived from the gene structure (exon-intron arrangement) to infer a new lipocalin phylogeny. We also present the novel gene structure of three insect lipocalins. The position and phase of introns are well preserved among lipocalin clades when mapped onto a protein sequence alignment, suggesting the homologous nature of these introns. Because of this homology, we use the intron position and phase of 23 lipocalin genes to reconstruct a phylogeny by maximum parsimony and distance methods. These phylogenies are very similar to the phylogenies derived from protein sequence. This result is confirmed by congruence analysis, and a consensus tree shows the commonalities between the two source trees. Interestingly, the intron arrangement phylogeny shows that metazoan lipocalins have more introns than other eukaryotic lipocalins, and that intron gains have occurred in the C-termini of chordate lipocalins. We also analyze the relationship of intron arrangement and protein tertiary structure, as well as the relationship of lipocalins with members of the proposed structural superfamily of calycins. Our congruence analysis validates the gene structure data as a source of phylogenetic information and helps to further refine our hypothesis on the evolutionary history of lipocalins.     

Birth-and-death evolution of the internalin multigene family in Listeria

The birth-and-death model of multigene family evolution describes patterns of gene origination, diversification and loss within multigene families. Since it was first developed in the 1990s, the model has been found to characterize a large number of eukaryotic multigene families. In this paper, we report for the first time a bacterial multigene family that undergoes birth-and-death evolution. By analyzing the evolutionary relationships among internalins, a relatively large and diverse family of genes associated with key virulence functions in Listeria, we demonstrate the importance of birth-and-death evolution in the diversification of this important bacterial pathogen. We also detected two instances of lateral gene transfer within the internalins, but the estimated frequency would have been much higher had it not been analyzed within the context of birth-and-death evolutionary dynamics and a phenomenon that we term "paralog-sorting", which involves the unequal transmittal of gene duplicates during or subsequent to the speciation process. As such, in addition to providing the first demonstration of birth-and-death evolution within a bacterial multigene family, our results indicate that the extent of lateral transfer in bacterial multigene families should be re-examined in the light of birth-and-death evolution.     

The structure and evolution of the human salivary proline-rich protein gene family

We present the nucleotide sequences of four members of the six-member human salivary prolinerich protein (PRP) gene family. The four genes are PRB1 and PRB2, which encode basic PRPs, and PRB3 and PRB4, which encode glycosylated PRPs. Each PRB gene is approximately 4.0 kb in length and contains four exons, the third of which is entirely composed of 63-bp tandem repeats and encodes the proline-rich portion of the protein products. Exon 3 contains different numbers of tandem repeats in the different PRB genes. Variation in the numbers of these repeats is also responsible for length variations in different alleles of the PRB genes. We have determined a probable evolutionary history of the human PRP gene family by comparing the nucleotide sequences of the six PRP genes. The present-day six PRP loci probably evolved from a single ancestral gene by four sequential gene duplications, leading to six genes that fall into three subsets, each consisting of two genes. During this evolutionary process, multiple rearrangements and gene conversion occurred mainly in the region from the 3 end of IVS2 and the 3 end of exon 3.     

Evolution of the casein multigene family: conserved sequences in the 5'' flanking and exon regions.

The rat alpha- and bovine alpha s1-casein genes have been isolated and their 5' sequences determined. The rat alpha-, beta-, gamma- and bovine alpha s1-casein genes contain similar 5' exon arrangements in which the 5' noncoding, signal peptide and casein kinase phosphorylation sequences are each encoded by separate exons. These findings support the hypothesis that during evolution, the family of casein genes arose by a process involving exon recruitment followed by intragenic and intergenic duplication of a primordial gene. Several highly conserved regions in the first 200 base pairs of the 5' flanking DNA have been identified. Additional sequence homology extending up to 550 base pairs upstream of the CAP site has been found between the rat alpha- and bovine alpha s1-casein sequences. Unexpectedly, the 5' flanking promoter regions are conserved to a greater extent than both the entire mature coding and intron regions of these genes. These conserved 5' flanking sequences may contain potential cis regulatory elements which are responsible for the coordinate expression of the functionally-related casein genes during mammary gland development.     

Molecular evolution of the Metazoan protein kinase C multigene family

Protein kinases C (PKCs) comprise closely related Ser/Thr kinases, ubiquitously present in animal tissues; they respond to second messengers, e.g., Ca²⁺ and/or diacylglycerol, to express their activities. Two PKCs have been sequenced from Geodia cydonium, a member of the lowest multicellular animals, the sponges (Porifera). One sponge G. cydonium PKC, GCPKC1, belongs to the ``novel' (Ca<sup>2+</sup>-independent) PKC (nPKC) subfamily while the second one, GCPKC2, has the hallmarks of the ``conventional' (Ca²⁺-dependent) PKC (cPKC) subfamily. The alignment of the Ser/Thr catalytic kinase domains, of the predicted aa sequences for these cDNAs with respective segments from previously reported sequences, revealed highest homology to PKCs from animals but also distant relationships to Ser/Thr kinases from protozoa, plants, and bacteria. However, a comparison of the complete structures of the sponge PKCs, which are—already—identical to those of nPKCs and cPKCs from higher metazoa, with the structures of protozoan, plant, and bacterial Ser/Thr kinases indicates that the metazoan PKCs have to be distinguished from the nonmetazoan enzymes. These data indicate that metazoan PKCs have a universal common ancestor which they share with the nonmetazoan Ser/Thr kinases with respect to the kinase domain, but they differ from them in overall structural composition. Received: 10 January 1996 / Accepted: 12 March 1996